Structures of Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) Isoforms and Their Interactions with Chloroquine

Using a recently elucidated atomic-resolution cryogenic electron microscopy (cryo-EM) structure for the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein 7G8 isoform as template [KimJ.; Nature2019, 576, 315−32031776516 ], we use Monte Carlo molecular dynamics (MC/MD) simulations of PfCRT embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane to solve energy-minimized structures for 7G8 PfCRT and two additional PfCRT isoforms that harbor 5 or 7 amino acid substitutions relative to 7G8 PfCRT. Guided by drug binding previously defined using chloroquine (CQ) photoaffinity probe labeling, we also use MC/MD energy minimization to elucidate likely CQ binding geometries for the three membrane-embedded isoforms. We inventory salt bridges and hydrogen bonds in these structures and summarize how the limited changes in primary sequence subtly perturb local PfCRT isoform structure. In addition, we use the “AlphaFold” artificial intelligence AlphaFold2 (AF2) algorithm to solve for domain structure that was not resolved in the previously reported 7G8 PfCRT cryo-EM structure, and perform MC/MD energy minimization for the membrane-embedded AF2 structures of all three PfCRT isoforms. We compare energy-minimized structures generated using cryo-EM vs AF2 templates. The results suggest how amino acid substitutions in drug resistance-associated isoforms of PfCRT influence PfCRT structure and CQ transport.


SUPPLEMENTAL FIGURES
Figure S1.Flowchart of computational work.A) The 7G8 EM (PBD 6UKJ) structure is imported into Maestro.B) Mutations are made in silico to generate additional HB3 and Dd2 isoform primary sequence, and protein structure for all membrane imbedded isoforms is prepared for MC/MD energy minimization (see methods).C) Energy minimized structures are generated for all isoforms by MC/MD and all frames from three independent simulations are clustered.D) The averaged structures are analyzed and salt bridges (SB) and hydrogen bonds (HB) are inventoried as described in methods.E) Structures are docked to CQ 2+ .F) The two highest ranked (see "drug docking", Methods) drug-poses are energy minimized as in B,C.G) docked structures are analyzed for protein-drug interactions (see text).H) AF2 structures are generated for HB3, 7G8, and Dd2 PfCRT isoform using isoform primary sequence.I) The AlphaFold (AF2) structures are membrane imbedded and prepared for MC/MD energy minimization as in "B" (see methods).J) The AF2 energy minimized structures are solved by MC/MD (as in "C") and all frames clustered to generate the presented "AFMD" structures.

Figure S2
Each MC/MD simulation"converges" (reaches a plateau of intrinsic random motion) after approximately 2 ns (red vertical bar), and beginning at 4 ns (the blue vertical bar) the protein isoform structures do not undergo any significant conformational change during further energy minimization.Data shown are one of three independent 10 ns MC/MD simulations for each PfCRT isoform; HB3 (blue, top), 7G8 (green, middle), and Dd2 (red, bottom).Note the plots have been offset from one another (-0.5 Å for 7G8 and -1.0 Å for Dd2, relative to HB3, c.f. Y axis) for ease of visual comparison.S5) are 424 residues +/-1 residue except for P. relictum CRT (Prel, 7 from bottom) which is listed as 414 residues, perhaps an artifactual result of a partial clone sequence since all residues missing vs all other CRTs are sequential and C terminal (denoted "-", Prel 413 -425 relative to Pfal).Black dots indicate PfCRT codon numbers which are found above the black dots, and horizontal lines above the sequences denote previously determined (black; see also Kim et al. 19 ) or AFMD resolved herein (orange) conformation and topology (see also text and Fig. S7).Colored circles above a given codon indicates the type of interaction in which the residue participates (see also tables S2, S3, S4); red indicates a SB; blue indicates a sidechain -sidechain HB; and green indicates a sidechainpeptide back bone HB, note some residues may participate in more than one type of interaction during protein conformational dynamics (compare tables S2 -S4).When the residue for a given interaction is identical across all 24 orthologues, it is indicated by a double asterisk "⁑" underneath the column of residues at a given position, while residues that are conserved in ≥ 22 orthologues are indicated by a single asterisk "*".Accession numbers can be found in [42].
Supplemental Tables Table S1.Amino acid differences between the three PfCRT isoforms studied in this paper.Highlighted in green are residues that differ for 7G8 and Dd2 (CQR) vs HB3 (CQS) isoforms.
Table S2.Lifetimes for all SB found at ≤4 Å hetero atom to hetero atom distance for ≥ 10 % of simulation time for at least one isoform (see text).The salt bridges are listed in order of the lowest numbered residue and the residue pairs are in the order of "donor"/"acceptor".Residues that are mutated in different isoforms are italicized.Color code denotes lifetime frequency (> 40%, green; 10-40%, yellow; <10%, red).Table S3.Lifetimes for all HB found at ≤3.2 Å hetero atom to hetero atom distance for ≥ 10 % of simulation time for at least one isoform (see text).The HB are listed in the order of the lowest numbered residue and residue pairs in the order of "HB donor"/"acceptor".Residues that are mutated in different isoforms are italicized.Color code denotes lifetime frequency (>50%, green; 10-50%, yellow; <10%, red).

Figure S7 .
Figure S7.Revised cartoon of PfCRT topology colored as in Kim et al. 19 and including a 3 helix "zipper" defined here based on PfCRT AFMD structures.Shown are side (left) or top down views (from the cytosol; right).The 10 TM core helices are numbered 1-10, along with portions of juxta membrane helix 1 (JM1; dark purple & orange) and JM2 (yellow -green), which were previously resolved in Kim et al 19 .Additional JM1 structure elucidated here via AF2 (see text) is shown in orange.The cytosolic zipper, consisting of newly defined JM0 (purple), JM1 (dark purple & orange), and JM3 (red) appears to fold directly above the cytosolic opening of the PfCRT central pore after MC/MD energy minimization (see also Fig. 8)

Table S4 .
Lifetimes for all side chain-backbone peptide bond HB found at ≤ 3.2 Å hetero atom to hetero atom distance for ≥ 10 % of simulation time for at least one isoform (see text).The HB are listed in the order of the lowest numbered residue and residue pairs are in the order of "HB donor"/"acceptor".Residues that are mutated in different isoforms are italicized.The residue immediately preceding the relevant peptide backbone is indicated in bold.Color code denotes lifetime frequency (> 50%, green; 10-50%, yellow; <10%, red).